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Abstract:

The present invention discloses for the first time that the insulin
receptor (IR) is a target of Herstatin, which modulates IR and
IR-mediated intracellular signaling. In preferred aspects, Herstatin
binds at nM concentrations to cell-surface IR, up-regulates basal IR
expression by several-fold, induces the accumulation of pro-IR, and
stimulates insulin activation of the ERK pathway. Moreover, these changes
in insulin signaling are accompanied by alterations in IGF-IR expression,
IRS-2 levels, and the serine phosphorylation state of both IRS-1 and
IRS-2. Preferred aspects provide novel therapeutic methods and
pharmaceutical compositions for treatment of conditions associated with
altered IR expression or IR-mediated signaling, including but not limited
to insulin resistance syndrome, pre-diabetic conditions, metabolic
syndrome, type 1 and type 2 diabetes, cardiac disease,
diabetes-associated vascular disease, atherosclerosis, hypertension,
diabetes-associated lipid metabolism disorders (dyslipidemia), obesity,
critical illness, neurodegenerative disorders, and combinations thereof,
and cancer.

Claims:

1. A method for treating a condition associated with altered insulin
receptor expression or altered insulin receptor-mediated signaling, said
method comprising administering to a subject in need thereof, a
therapeutically effective amount of Herstatin, or a variant thereof, that
binds to the insulin receptor.

2. A method for treating a condition associated with altered insulin
receptor expression or altered insulin receptor-mediated signaling,
comprising administering to a subject in need thereof, a therapeutically
effective amount of a Int8 RBD polypeptide, or a variant thereof, that
binds to the insulin receptor.

4. The method of any one of claims 1 or 2, wherein the cell further
expresses at least one target receptor selected from the group consisting
of: EGFR (HER-1, erbB-1); ΔEGFR; HER-2 (erbB-2); HER-3 (erbB-3);
HER-4 (erbB-4); and IGF-IR.

5. The method of claim 1, wherein the Herstatin, or variant thereof,
comprises a polypeptide selected from the group consisting of SEQ ID
NO:2, or a fragment of SEQ ID NO:2 of about 80 to 419 contiguous residues
in length, wherein the C-terminal 79 contiguous amino acids are present,
wherein at least one N-linked glycosylation site is present, and wherein
the polypeptide binds to the insulin receptor.

6. The method of claim 1, wherein the Herstatin, or variant thereof,
comprises a sequence selected from the group consisting of SEQ ID
NOS:32-42.

8. The method of claim 2, wherein the Int8 RBD polypeptide, or a variant
thereof comprises a polypeptide selected from the group consisting of SEQ
ID NO:1, or a fragment of SEQ ID NO:1 of about 50 to 79 contiguous
residues in length, wherein the polypeptide binds to the insulin
receptor.

9. The method of claim 2, wherein the Int8 RBD polypeptide, or a variant
thereof, comprises a sequence selected from the group consisting of SEQ
ID NOS:21-31,

11. The method of any one of claims 1 or 2, further comprising
administering a therapeutically effective amount of a receptor-specific
antibody that binds to a target receptor selected from the group
consisting of: insulin receptor (IR), EGFR (HER-1, erbB-1); ΔEGFR;
HER-2 (erbB-2); HER-3 (erbB-3); HER-4 (erbB-4), and IGF-IR.

12. The method of any one of claims 1 or 2, further comprising
administration of a therapeutically effective amount of an agent selected
from the group consisting of: insulin, insulin-sensitizing agents,
insulin secretogogues, and combinations thereof.

13. The method of claim 12, wherein the insulin-sensitizing agent is
selected from the group consisting of biguanides, metformin,
thiazolidinediones (glitazones), and combinations thereof.

14. The method of claim 12, wherein the insulin secretogogue is selected
from the group consisting of sulfonylureas, meglitinides, and
combinations thereof.

15. A pharmaceutical composition for treating a condition associated with
altered insulin receptor expression or altered insulin receptor-mediated
signaling, comprising, Herstatin, or a variant thereof, that binds to the
insulin receptor and a pharmaceutically acceptable carrier or excipient.

16. A pharmaceutical composition for treating a condition associated with
altered insulin receptor expression or altered insulin receptor-mediated
signaling, comprising, a Int8 RBD polypeptide, or a variant thereof, that
binds to the insulin receptor and a pharmaceutically acceptable carrier
or excipient.

18. The pharmaceutical composition of claim 15, wherein the Herstatin, or
variant thereof, comprises a polypeptide selected from the group
consisting of SEQ ID NO:2, or a fragment of SEQ ID NO:2 of about 80 to
419 contiguous residues in length, wherein the C-terminal 79 contiguous
amino acids are present, wherein at least one N-linked glycosylation site
is present, and wherein the polypeptide binds to the insulin receptor.

19. The pharmaceutical composition of claim 16, wherein the Int8 RBD
polypeptide, or a variant thereof comprises a polypeptide selected from
the group consisting of SEQ ID NO:1, or a fragment of SEQ ID NO:1 of
about 50 to 79 contiguous residues in length, wherein the polypeptide
binds to the insulin receptor.

20. The pharmaceutical composition of any one of claims 15 or 16, further
comprising an agent selected from the group consisting of: insulin,
insulin-sensitizing agents, insulin secretogogues, and combinations
thereof.

21. The pharmaceutical composition of claim 20, wherein the
insulin-sensitizing agent is selected from the group consisting of
biguanides, metformin, thiazolidinediones (glitazones), and combinations
thereof.

22. The pharmaceutical composition of claim 20, wherein the insulin
secretogogue is selected from the group consisting of sulfonylureas,
meglitinides, and combinations thereof.

23. A method for targeting a therapeutic agent to a cell expressing
insulin receptor, comprising attaching the therapeutic agent to
Herstatin, or to a variant thereof, that binds to the extracellular
domain of a cellular target insulin receptor.

24. A method for targeting a therapeutic agent to a cell expressing
insulin receptor, comprising attaching the therapeutic agent to a Int8
RBD polypeptide, or a variant thereof, that binds to the cellular target
insulin receptor.

25. The method of any one of claims 23 or 24, wherein the cell further
expresses a target receptor selected from the group consisting of: EGFR
(HER-1, erbB-1); ΔEGFR; HER-2 (erbB-2); HER-3 (erbB-3); HER-4
(erbB-4); IGF-IR, and combinations thereof.

26. The method of claim 23, wherein the wherein the Herstatin, or variant
thereof, comprises a polypeptide selected from the group consisting of
SEQ ID NO:2, or a fragment of SEQ ID NO:2 of about 80 to 419 contiguous
residues in length, wherein the C-terminal 79 contiguous amino acids are
present, wherein at least one N-linked glycosylation site is present, and
wherein the polypeptide binds to the insulin receptor.

27. The method of claim 24, wherein the Int8 RBD polypeptide, or a variant
thereof comprises a polypeptide selected from the group consisting of SEQ
ID NO:1, or a fragment of SEQ ID NO:1 of about 50 to 79 contiguous
residues in length, wherein the polypeptide binds to the insulin
receptor.

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001]This application claims the benefit of priority to: U.S. Provisional
Patent Application Ser. No. 60/616,596, filed 5 Oct. 2004 and entitled
"COMPOSITIONS AND METHODS FOR TREATING DISEASE"; and to U.S. Provisional
Patent Application Ser. No. 60/688,355, filed 6 Jun. 2005, of same title,
both of which are incorporated by reference herein in their entireties.

FIELD OF THE INVENTION

[0002]Aspects of the invention relate generally to therapeutic molecules,
compositions and methods for treatment of diseases through modulation of
the insulin receptor (IR) and IR-mediated intracellular signaling by
administration of Herstatin or variants thereof, and in more particular
aspects relate to compositions and methods for cell targeting, and for
the treatment of conditions or diseases associated with altered IR
expression or altered IR-mediated signaling, including but not limited to
insulin resistance syndrome, pre-diabetic conditions, metabolic syndrome,
type 1 and type 2 diabetes, cardiac disease, diabetes-associated vascular
disease, atherosclerosis, hypertension, diabetes-associated lipid
metabolism disorders (dyslipidemia), obesity, critical illness,
neurodegenerative disorders, and combinations thereof, and cancer.

BACKGROUND

[0003]The Insulin Receptor. The insulin receptor is the canonical member
of the insulin receptor family of receptor tyrosine kinases, which also
includes the IGF-IR and the insulin receptor-related receptor (IRR).
These molecules share a heterotetrameric structure comprised of two
extracellular ligand-binding α subunits, which are coupled to each
other and to two transmembrane β subunits by disulfide linkages. The
intracellular portion of the β subunit contains the intrinsic
tyrosine kinase catalytic domain, which is activated by binding of
extracellular ligand and a presumed conformational change in the β
subunit. The activated receptor undergoes autophosphorylation of tyrosine
residues in the kinase domain as well as residues in the flanking
juxtamembrane and carboxyl-terminal domains. The phosphorylation of these
residues, particularly in the juxtamembrane region, allows the
recruitment of scaffolding adapter proteins such as IRS-1 and IRS-2 and
Shc, which are then phosphorylated on tyrosine residues by the activated
receptor to recruit a second level of signaling molecules to initiate the
signaling cascades that are responsible for insulin action. These include
the ERK arm of the MAPK pathway, the P13K-Akt/PKB pathway, and the
APS-Cbl-CrkII-TC10 pathway. In cells expressing both insulin and IGF-I
receptors, hybrid receptors consisting of insulin and IGF-I receptor
α-β hemireceptors can form. These are activated by IGF-I but
not by insulin. The insulin receptor family of receptors differs from the
erbB/Her receptors by virtue of their existence as pre-dimerized
heterotetramers and their use of intermediates such as IRS and Shc
proteins to couple to downstream signaling pathways.

[0004]Diabetes and Related Conditions. The epidemic of obesity occurring
in the in the United States and around the world portends a significant
increase in type 2 diabetes mellitus in the adult and, increasingly, in
the pediatric populations. There is also growing concern regarding the
prevalence of pre-diabetic conditions such as the metabolic syndrome, the
incidence of which dwarfs that of clinically apparent diabetes per se.
The hyperglycemia of type 2 diabetes results from defects in both insulin
sensitivity and pancreatic β-cell function, leading to a relatively
insulin-deficient state. There is also a growing appreciation that
insulin resistance may play an important role in cardiac disease. A
mainstay of current therapy is the use of insulin-sensitizing agents such
as metformin and thiazolidinediones that act to enhance the ability of
insulin to trigger appropriate cellular responses such as glucose
transport in insulin target tissues. These treatments suffer, however,
from a lack of mechanistic specificity, high. rates of unresponsiveness
(up to 30% for thiazolidinediones), and frequent side effects. Although
advances are being made in the generation of islets for transplant, the
time frame for the successful application of these approaches in human
patients with both type 1 and type 2 disease and their ability to affect
insulin resistance remains unclear. Thus, there continues to be an urgent
need to design new and novel therapies to treat insulin resistance (see,
e.g., Alsheikh-Ali & Karas, Amer J Cardiology, 93:1417-8, 2004; Ovalle &
Fernando, Southern Med J., 95:1188-94, 2002; and Zangeneh et al., Mayo
Clinic Proc. 78:471-479, 2003).).

[0009]There is, therefore, a need in the art to further investigate and
characterize the interactions among the IR, the erbB family receptors,
and the IGF-I receptor, and to identify modulators of the signaling
mediated by these receptors.

[0010]There is a pronounced need in the art to identify and develop IR
modulators as therapeutic agents.

[0011]There is a pronounced need in the art to design new and novel
therapies to treat insulin resistance.

[0012]There is a need in the art to further assess and exploit the
receptor-modulating utilities of Herstatin.

SUMMARY OF THE INVENTION

[0013]The present invention relates to therapeutic molecules and
compositions for modulation of the insulin receptor (IR) and IR-mediated
intracellular signaling by administration of an isoform of a cell surface
receptor, and in preferred aspects, to administration of Herstatin, which
is an example of such a cell surface receptor isoform. Aspects of the
invention are based upon the discovery that the insulin receptor (IR) is
a target of Herstatin, which specifically binds to the IR with nM
affinity. According to preferred aspects of the present invention,
Herstatin alters the landscape of IR-mediated signaling, exerting a
positive effect on IR expression, and substantially increasing
IR-mediated ERK pathway activation. The MEK (MAPK kinase)-ERK pathway has
been shown to be significantly involved in glucose transport (e.g.,
Harmon et al., Am. J. Physiol. Endocrinol. Metab., 287:E758-E766, 2004).

[0014]In particular aspects, Herstatin was shown herein to bind at nM
concentrations to cell-surface IR, to up-regulate basal IR expression by
several-fold, and to induce the accumulation of pro-IR.

[0015]In additional aspects, and with respect to signal transduction,
Herstatin was shown herein to substantially (e.g., >40-fold) stimulate
insulin activation of the ERK pathway, but to have little effect on
insulin-stimulated activation of the P13K/Akt pathway.

[0016]In further aspects, these changes in insulin signaling were shown
herein to be accompanied by about a 4-fold decrease in IGF-IR expression,
a decrease in the apparent serine phosphorylation state of IRS-1, and a
slight decrease in IRS-2 levels as well as a decrease in apparent serine
phosphorylation of IRS-2.

[0017]Therefore, according to particular aspects of the present invention,
Herstatin, a cell surface receptor isoform, has substantial utility for
modulating insulin signaling in cells expressing IR.

[0019]Alternative preferred aspects provide for a novel use of Herstatin
in therapeutic methods and pharmaceutical compositions for treating
various diseases associated with or characterized by alterations in
insulin sensitivity or resistance (e.g., conditions or diseases
characterized by altered IR expression and/or altered IR-related
signaling).

[0022]Methods of treatment. Particularly preferred embodiments provide a
method for treating or modulating a condition having an aspect related
to, or associated with, or characterized by altered IR expression or
altered IR-mediated signaling at a cellular level, comprising
administering to a subject having such a condition, a therapeutically
effective amount of a cell surface receptor isoform such as Herstatin, or
a variant thereof (e.g., a therapeutically effective amount of a Int8 RBD
polypeptide, or a variant thereof), that binds to the extracellular
domain of cellular target IR. Preferably, the condition is selected from
the group consisting of insulin resistance, pre-diabetic conditions,
metabolic syndrome, type 1 and type 2 diabetes, cardiac disease,
diabetes-associated vascular disease, diabetes-associated lipid
metabolism disorders, neurodegenerative disorders, and combinations
thereof. In alternative related embodiments, the cell further expresses a
target receptor selected from the group consisting of: EGFR (HER-1,
erbB-1); ΔEGFR; HER-2 (erbB-2); HER-3 (erbB-3); HER-4 (erbB-4);
IGF-IR and combinations thereof.

[0023]Alternative related preferred embodiments further comprise
administering a therapeutically effective amount of a molecule such as a
small molecule, protein, peptide or receptor-specific antibody that binds
to the extracellular domain of a target receptor selected from the group
consisting of: IR, EGFR (HER-1, erbB-1); ΔEGFR; HER-2 (erbB-2);
HER-3 (erbB-3); HER-4 (erbB-4), and IGF-IR.

[0024]Preferably, the methods further comprise administration of the cell
surface receptor isoforms of this invention in combination with a
therapeutically effective amount of an agent selected from the group
consisting of: insulin, insulin-sensitizing agents, insulin
secretogogues, and combinations thereof. Preferably, the
insulin-sensitizing agent is selected from the group consisting of
biguanides, metformin, thiazolidinediones (glitazones), and combinations
thereof. Preferably, the insulin secretogogue is selected from the group
consisting of sulfonylureas, meglitinides, and combinations thereof.

[0025]Pharmaceutical compositions. Additional preferred embodiments
provide a pharmaceutical composition for treating a condition having an
aspect related to, or associated with or characterized by altered IR
expression or altered IR-mediated signaling at a cellular level,
comprising, along with a pharmaceutically acceptable carrier or
excipient, a cell surface receptor isoform such as Herstatin, or a
variant thereof (e.g., a Int8 RBD polypeptide, or a variant thereof),
that binds to the extracellular domain of a cellular target IR.
Preferably, the condition is selected from the group consisting of
insulin resistance syndrome, pre-diabetic conditions, metabolic syndrome,
type 1 and type 2 diabetes, cardiac disease, diabetes-associated vascular
disease, atherosclerosis, hypertension, diabetes-associated lipid
metabolism disorders (dyslipidemia), obesity, critical illness,
neurodegenerative disorders, and combinations thereof. In alternative
related preferred embodiments, the targeted cell further expresses a
target receptor selected from the group consisting of: EGFR (HER-1,
erbB-1); ΔEGFR; HER-2 (erbB-2); HER-3 (erbB-3); HER-4 (erbB-4);
IGF-IR, and combinations thereof. Preferably, the pharmaceutical
composition further comprises an agent selected from the group consisting
of: insulin, insulin-sensitizing agents, insulin secretogogues, and
combinations thereof Preferably, the insulin-sensitizing agent is
selected from the group consisting of biguanides, metformin,
thiazolidinediones (glitazones), and combinations thereof. Preferably,
the insulin secretogogue is selected from the group consisting of
sulfonylureas, meglitinides, and combinations thereof.

[0026]Cell targeting. Yet further preferred embodiments provide methods
and compositions for targeting a therapeutic agent to a cell expressing
IR, comprising attaching the therapeutic agent to the cell surface
receptor isoform, such as Herstatin, or to a variant thereof (e.g., a
Int8 RBD polypeptide, or a variant thereof), that binds to the
extracellular domain of a cellular target IR.

[0028]Preferably, in all of the above-described preferred embodiments, the
Herstatin, or variant thereof, comprises a polypeptide selected from the
group consisting of SEQ ID NO:2, or a fragment of SEQ ID NO:2 of about 80
to 419 contiguous residues in length, wherein the C-terminal 79
contiguous amino acids are present, wherein at least one N-linked
glycosylation site is present, and wherein the polypeptide binds to the
extracellular domain of insulin receptor with an affinity binding
constant of at least 108 M-1. In particular aspects, the
Herstatin, or variant thereof, comprises a sequence selected from the
group consisting of SEQ ID NOS:32-42. Preferably, the Herstatin or
variant thereof comprises SEQ ID NO:32. Preferably, the Herstatin or
variant thereof consists of SEQ ID NO:32.

[0029]Preferably, the Int8 RBD polypeptide, or a variant thereof comprises
a polypeptide selected from the group consisting of SEQ ID NO:1, or a
fragment of SEQ ID NO:1 of about 50 to 79 contiguous residues in length,
wherein the polypeptide binds to the extracellular domain of insulin
receptor with an affinity binding constant of at least 108 M-1.
In particular aspects, the Int8 RBD polypeptide, or a variant thereof,
comprises a sequence selected from the group consisting of SEQ ID
NOS:21-31. Preferably, the Int8 RBD polypeptide or variant thereof
comprises SEQ ID NO:21. Preferably, the Int8 RBD polypeptide or variant
thereof consists of SEQ ID NO:21.

BRIEF DESCRIPTION OF THE DRAWINGS

[0030]FIG. 1 shows, according to particular aspects of the present
invention as described in more detail in EXAMPLE II below, that Herstatin
bound at nM concentrations to 3T3 cells over-expressing insulin receptor
(IR), but not to 3T3 parental cells.

[0031]FIGS. 2A and 2B show, according to particular aspects as described
in more detail in EXAMPLE III below, that Herstatin expression
up-regulated IR expression and activation in MCF-7 cells.

[0032]FIGS. 3A and 3B show, according to particular aspects as described
in more detail in EXAMPLE IV below, that in MCF-7 cells Herstatin
expression substantially amplified insulin-stimulated ERK activation.

[0033]FIGS. 4A, 4B, 4C and 4D show, according to particular aspects as
described in more detail in EXAMPLE V below, that Herstatin altered the
expression of an array of proteins that are directly involved in insulin
action.

[0034]FIG. 5 shows, according to particular aspects, that the EGFR
inhibitor AS1478 does not affect insulin signaling.

[0035]FIG. 6 shows, according to particular aspects, that inhibition of
the EGF receptor with an EGF receptor-specific inhibitor does not lead to
an increase in insulin receptor.

DETAILED DESCRIPTION OF THE INVENTION

[0036]Herstatin is an example of a cell surface receptor isoform, that may
also be referred to as an alternative receptor product or an intron
fusion protein, which functions as a receptor ligand, and functions as a
secreted ligand that inhibits members of the EGF receptor family.
Herstatin binds with high affinity to all members of the EGF receptor
family, including EGFR/HER1/erbB1, HER2/neu/erbB2, HER3/erbB3,
HER4/erbB4, and to ΔEGFR, and further binds to the IGF-IR.

[0037]The present invention discloses for the first time that the insulin
receptor (IR) is a target of the cell surface receptor isoform,
Herstatin, which specifically binds to the IR with nM affinity. According
to preferred aspects of the present invention, Herstatin binds at nM
concentrations to cell-surface IR, and further modulates insulin
signaling in cells (e.g., MCF-7 human breast cancer cells, etc)
expressing IR.

[0038]Herstatin is disclosed herein to alter expression of the IR and in
particular to up-regulate basal IR expression by several-fold, and induce
the accumulation of pro-IR.

[0039]Herstatin is further disclosed herein to modulate insulin
activation. Herstatin stimulates insulin activation of the ERK pathway in
a range of about 5- to about 80-fold, while having a more modest to
little effect on insulin-stimulated (IR-mediated) activation of the
P13K/Akt pathway.

[0040]Significantly, these changes in insulin signaling were shown herein
to be accompanied by a decrease in IGF-IR expression in the range of
about a 2- to about a 10-fold decrease, a decrease in the apparent serine
phosphorylation state of IRS-1, and a slight decrease in IRS-2 levels as
well as a decrease in apparent serine phosphorylation of IRS-2.

[0041]Therefore, preferred aspects of the present invention provide for
uses of Herstatin in novel methods and compositions for treating a
condition having an aspect related to, or associated with or
characterized by altered IR expression or IR-mediated signal
transduction.

[0042]The instant description and Examples, in various aspects, disclose
the ability of Herstatin to modulate insulin action in cell models (e.g.,
a breast cancer cell model that consists of the well-characterized MCF-7
human breast cancer cell line, and two derivative clones that express
human Herstatin from a stably transfected expression vector).

[0043]In particular aspects, Herstatin binding to cell-surface IR was
investigated using IR-expressing 3T3 cells (IRA-3T3). Moreover, the
effects of Herstatin on the expression and activation of the IR itself,
and upon the expression and activation of the major signaling pathways
that emanate from the activated insulin receptor (e.g., the ERK pathway
and the P13K/Akt pathway) were investigated in MCF-7 and in
Herstatin-expressing MCF-7 cells. All of the individual assays were
repeated a minimum of three times with similar, if not identical,
results, and many of the findings have been replicated and confirmed in
experiments with an independent Herstatin-expressing MCF-7 clone.

[0048]As used herein, an isoform of a cell surface receptor (also referred
to herein as a CSR isoform), such as an isoform of a receptor tyrosine
kinase, refers to a receptor that lacks a domain or portion thereof
sufficient to alter or modulate a biological activity of the receptor or
modulate a biological activity compared to a wildtype and/or predominant
form of the receptor. A CSR isoform refers to a receptor that lacks a
domain or portion of a domain sufficient to alter or modulate a
biological activity of the receptor, for example the insulin receptor.
Generally, a biological activity is altered in an isoform at least 0.1,
0.5, 1, 2, 3, 4, 5, or 10-fold compared to a wildtype and/or predominant
form of the receptor. Typically, a biological activity is altered 10-,
20-, 50-, 100- or 1000-fold or more. With reference to an isoform,
alteration of activity refers to difference in activity between the
particular isoform, which is shortened, compared to the unshortened form
of the receptor. Alteration of biological activity includes an
enhancement or a reduction of activity. In particular embodiments,
alteration of a biological activity is a reduction in the activity. In
particular embodiments, an alteration of a biological activity is a
reduction in biological activity, and the reduction can be at least 0.1
0.5 1, 2, 3, 4, 5, or 10-fold compared to a wildtype and/or predominant
form of the receptor. Typically, a biological activity is reduced 5, 10,
20, 50, 100 or 1000-fold or more. Reference herein to a CSR isoform with
altered activity refers to the alteration in an activity by virtue of the
different structure or sequence of the CSR isoform compared to a cognate
receptor.

[0049]Reference herein to modulating the activity of a target cell surface
receptor means that a CSR isoform interacts in some manner with the
target receptor and activity, such as ligand binding or dimerization or
other signal-transduction-related activity is altered.

[0050]Intron fusion proteins (IFPs) are exemplary CSR isoforms. IFPs, for
purposes herein include natural and combinatorial IFPs. A natural IFP
refers to a polypeptide that is encoded by an alternatively spliced RNA
that contains one or more amino acids encoded by an intron operatively
linked to one or more portions of the polypeptide encoded by one or more
exons of a gene. Alternatively spliced mRNA is one that is isolated or is
one that can be prepared synthetically by joining splice donor and
acceptor sites in a gene. A natural IFP contains one or more amino acids
and/or one or more stop codons encoded by an intron sequence. A
combinatorial IFP refers to a polypeptide that is shortened compared to a
wildtype or predominant form of a polypeptide. Typically, the shortening
removes one or more domains or a portion thereof from a polypeptide such
that a biological activity is altered. Combinatorial IFPs often mimic a
natural IFP in that one or more domains or a portion thereof that is/are
deleted in a natural IFP derived from the same gene sequence or derived
from a gene sequence in a related gene family.

[0051]As used herein, natural with reference to IFP, refers to any
protein, polypeptide or peptide or fragment thereof (by virtue of the
presence of the appropriate splice acceptor/donor sites) that is encoded
within the genome of an animal and/or is produced or generated in an
animal or that could be produced from a gene. Natural IFPs include
allelic variant. IFPs can be modified post-translationally.

[0058]The phrase "mutant form of HER-3" refers to a HER-3 protein having a
substitution of Glu for Gly in the ectodomain of HER-3 corresponding to a
single point mutation at nucleotide position 1877 ("a" instead of "g" at
this position), resulting in substitution of Glu instead of Gly at
residue position 560) (cDNA: SEQ ID NO:13; protein: SEQ ID NO:14).

[0062]Provided herein are cell surface receptor (CSR) isoforms (including
intron fusion proteins; IFPs) having the novel biological activity of
altering IR expression or altered IR mediated signaling. The CSR isoforms
differ from the cognate receptors in that there are insertions and/or
deletions, and the resulting CSR isoforms exhibit a difference in one or
more activities or functions compared to the cognate receptor. Such
differences include, for example elimination of all or part of a
transmembrane domain, and/or a change in a biological activity of the CSR
(e.g., as disclosed herein, the ability to modulate insulin receptor (IR)
expression or IR-mediated signaling). The CSR isoforms provided herein
can be used for modulating the activity of a cell surface receptor (e.g.,
the IR). They also can be used as targeting agents (e.g., targeting IR)
for delivery of molecules, such as drugs or toxins or nucleic acids, to
targeted cells or tissues.

[0063]A CSR isoform refers to a receptor that lacks a domain or portion of
a domain sufficient to alter a biological activity (e.g., an activity
with respect to the IR). Thus, an isoform may differ from a wildtype
and/or predominant form of the receptor, in that it lacks one or more
biological activities of the receptor. Additionally, CSR isoforms can
contain a new domain and/or biological function as compared to a wildtype
and/or predominant form of the receptor. For example, intron-encoded
amino acids can introduce a new domain or portion thereof into a CSR
isoform. Biological activities that can be altered (or gained) include,
but are not limited to, protein-protein interactions such as
dimerization, multimerization and complex formation, specificity and/or
affinity for ligand, cellular localization and relocalization, membrane
anchoring, enzymatic activity such as kinase activity, response to
regulatory molecules including regulatory proteins, cofactors, and other
signaling molecules, such as in a signal transduction pathway. Generally,
a biological activity is altered in an isoform at least 0.1, 0.5, 1, 2,
3, 4, 5, or 10-fold as compared to a wildtype and/or predominant form of
the receptor. Typically, a biological activity is altered 10, 20, 50, 100
or 1000-fold or more. For example, an isoform can be reduced with respect
to a particular biological activity.

[0064]CSR isoforms can also modulate an activity of a wildtype and/or
predominant form of the cognate receptor. For example, a CSR isoform can
interact directly or indirectly with a CSR isoform and modulate a
biological activity of the cognate receptor. Biological activities that
can be altered include, but are not limited to, protein-protein
interactions such as dimerization, multimerization and complex formation,
specificity and/or affinity for ligand, cellular localization and
relocalization, membrane anchoring, enzymatic activity such as kinase
activity, response to regulatory molecules including regulatory proteins,
cofactors, and other signaling molecules, such as in a signal
transduction pathway.

[0065]A CSR isoform can interact directly or indirectly with a cell
surface receptor to cause or participate in a biological effect, such as
by modulating a biological activity of the cell surface receptor (e.g.,
in the instant case, the IR). A CSR isoform also can interact
independently of a cell surface receptor to cause a biological effect,
such as by initiating or inhibiting a signal transduction pathway. For
example, a CSR isoform can initiate a signal transduction pathway and
enhance or promote cellular metabolism. In another example, a CSR isoform
can interact with the cell surface receptor as a ligand, causing a
biological effect for example by inhibiting a signal transduction pathway
that can promote or alter a cellular response to insulin. Hence, the
isoforms provided herein can function as cell surface receptor ligands in
that they interact with the targeted receptor in the same manner that a
cognate ligand interacts with and alters receptor activity. The isoforms
can bind as a ligand, but not necessarily to the ligand binding site, and
can serve to block receptor dimerization. They act as ligands in the
sense that they interact with the receptor. The CSR isoforms also can act
by binding to ligands for the receptor and/or by preventing receptor
activities, such as dimerization.

[0066]For example, a CSR isoform can compete with a CSR for ligand
binding. A CSR isoform can act as a dominant negative inhibitor, for
example, when complexed with a CSR. A CSR isoform can act as a dominant
negative inhibitor or as a competitive inhibitor of a CSR, for example,
by complexing with a CSR isoform and altering the ability of the CSR to
multimerize (e.g, dimerize or trimerize) with other CSRs. A CSR isoform
can compete with a CSR for interactions with other polypeptides and
cofactors in a signal transduction pathway.

[0067]The cell surface isoforms and families of isoforms provided herein
include, for example, isoforms of the HER-2 receptor (e.g., Herstatin),
IR, etc. Pharmaceutical compositions containing one or more different CSR
isoforms are provided. Also provided are methods of treatment of diseases
and conditions by administering the pharmaceutical compositions or
delivering a CSR isoform, such by administering the isoform protein
(polypeptide, etc), and/or by administration of a vector that encodes the
isoform. Administration, by either means, can be effected in vivo or ex
vivo. Also provided are methods for expressing, isolating and formulating
CSR isoforms.

Herstatin and/or RBD Int8 Polyepeptides and Therapeutic Agents

[0068]In preferred aspects, the present invention provides for Herstatin
(e.g., the sequences of SEQ ID NO:2) and polypeptides thereof that bind
to a insulin receptor (IR) as a target receptor (specifically, or in
addition to the known targets: EGFR, HER-2, HER-3, DEGFR, HER-4 and
IGF-IR). Also provided are RBD Int8 polypeptides (e.g., the sequences of
SEQ ID NO:1) and receptor-binding polypeptides thereof that bind to a
insulin receptor as a target receptor (specifically, or in addition to
the known targets EGFR, HER-2, HER-3, DEGFR, HER-4 and IGF-IR).

[0069]Preferably, the Herstatin and/or RBD Int8 polypeptides comprise an
amino acid sequence of SEQ ID NO:1 (or of SEQ ID NO:1 having from 1, to
about 3, to about 5, to about 10, or to about 20 conservative amino acid
substitutions), or a fragment of a sequence of SEQ ID NO:1 (or a fragment
of SEQ ID NO:1 having from 1, to about 3, to about 5, to about 10, or to
about 20 conservative amino acid substitutions) of about 50 to 79
contiguous residues in length, wherein the polypeptide binds to the
extracellular domain (ECD) of a target receptor (e.g., EGFR, HER-2,
HER-3, DEGFR, HER-4, IGF-IR and IR (as disclosed herein)) with an
affinity binding constant of at least 107 M-1, at least
5×107 M-1, or at least 108 M-1. Preferably, the
Herstatin and/or RBD Int8 polypeptide is from about 69 to 79 contiguous
residues in length, with a IR affinity binding constant of at least
107 M-1, at least 5×107 M-1, or at least
108 M-1 (similar to the respective binding constants associated
with the known EGFR, HER-2, HER-3, DEGFR, HER-4 and IGF-IR target
receptors). Preferably, Herstatin and/or RBD Int8 polypeptide comprises a
sequence of SEQ ID NO:1, or a conservative amino acid substitution
variant thereof. In particular aspects, the Int8 RBD polypeptide, or a
variant thereof, comprises a sequence selected from the group consisting
of SEQ ID NOS:21-31. Preferably, the Int8 RBD polypeptide or variant
thereof comprises SEQ ID NO:21. Preferably, the Int8 RBD polypeptide or
variant thereof consists of SEQ ID NO:21.

[0070]Preferably, the Herstatin and/or RBD Int8 polypeptides comprise an
amino acid sequence of SEQ ID NO:2 (or of SEQ ID NO:2 having from 1, to
about 3, to about 5, to about 10, or to about 20 conservative amino acid
substitutions), or a fragment of a sequence of SEQ ID NO:2 (or a fragment
of SEQ ID NO:2 having from 1, to about 3, to about 5, to about 10, or to
about 20 conservative amino acid substitutions) of about 80 to 419
contiguous residues in length, wherein the C-terminal 79 contiguous amino
acids are present, and wherein the polypeptide binds to the extracellular
domain (ECD) of a IR with an affinity binding constant of at least
107 M-1, at least 5×107 M-1, or at least
108 M-1 (similar to the respective binding constants associated
with the known EGFR, HER-2, HER-3, DEGFR, HER-4 and IGF-IR target
receptors). Preferably, the Herstatin and/or RBD Int8 polypeptide is from
about 350 to 419 contiguous residues in length, wherein the polypeptide
binds to the extracellular domain (ECD) of a IR with an affinity binding
constant of at least 107 M-1, at least 5×107
M-1, or at least 108 M-1 (similar to the respective
binding constants associated with the known EGFR, HER-2, HER-3, DEGFR,
HER-4 and IGF-IR target receptors). Preferably, comprises a sequence of
SEQ ID NO:2, or a conservative amino acid substitution variant thereof.
In particular aspects, the Herstatin, or variant thereof, comprises a
sequence selected from the group consisting of SEQ ID NOS:32-42.
Preferably, the Herstatin or variant thereof comprises SEQ ID NO:32.
Preferably, the Herstatin or variant thereof consists of SEQ ID NO:32.

Biologically Active Variants

[0071]Variants of Herstatin and/or RBD Int8 polypeptide have substantial
utility in various aspects of the present invention. Variants can be
naturally or non-naturally occurring. Naturally occurring variants are
found in humans or other species and comprise amino acid sequences which
are substantially identical to the amino acid sequences shown in SEQ ID
NO:1 or SEQ ID NO:2, and include natural sequence polymorphisms. Species
homologs of the protein can be obtained using subgenomic polynucleotides
of the invention, as described below, to make suitable probes or primers
for screening cDNA expression libraries from other species, such as mice,
monkeys, yeast, or bacteria, identifying cDNAs which encode homologs of
the protein, and expressing the cDNAs as is known in the art.

[0072]Non-naturally occurring variants which retain substantially the same
biological activities as naturally occurring protein variants, including
the target RBD activity and the modulation of target receptor signaling
activity, are also included here. Preferably, naturally or non-naturally
occurring variants have amino acid sequences which are at least 85%, 90%,
or 95% identical to the amino acid sequence shown in SEQ ID NOS:1 or 2.
More preferably, the molecules are at least 98% or 99% identical. Percent
identity is determined using any method known in the art. A non-limiting
example is the Smith-Waterman homology search algorithm using an affine
gap search with a gap open penalty of 12 and a gap extension penalty of
1. The Smith-Waterman homology search algorithm is taught in Smith and
Waterman, Adv. Appl. Math. 2:482-489, 1981.

[0073]As used herein, "amino acid residue" refers to an amino acid formed
upon chemical digestion (hydrolysis) of a polypeptide at its peptide
linkages. The amino acid residues described herein are generally in the
"L" isomeric form. Residues in the "D" isomeric form can be substituted
for any L-amino acid residue, as long as the desired functional property
is retained by the polypeptide. NH2 refers to the free amino group
present at the amino terminus of a polypeptide. COOH refers to the free
carboxy group present at the carboxyl terminus of a polypeptide. In
keeping with standard polypeptide nomenclature described in J. Biol.
Chem., 243:3552-59 (1969) and adopted at 37
C.F.R..§§.1.821-1.822, abbreviations for amino acid residues
are shown in Table 1:

[0074]It should be noted that all amino acid residue sequences represented
herein by a formula have a left to right orientation in the conventional
direction of amino-terminus to carboxyl-terminus. In addition, the phrase
"amino acid residue" is defined to include the amino acids listed in the
Table of Correspondence and modified and unusual amino acids, such as
those referred to in 37 C.F.R..§§ 1.821-1.822, and incorporated
herein by reference. Furthermore, it should be noted that a dash at the
beginning or end of an amino acid residue sequence indicates a peptide
bond to a further sequence of one or more amino acid residues or to an
amino-terminal group such as NH2 or to a carboxyl-terminal group
such as COOH.

[0075]Guidance in determining which amino acid residues can be
substituted, inserted, or deleted without abolishing biological or
immunological activity can be found using computer programs well known in
the art, such as DNASTAR® software. Preferably, amino acid changes in
the protein variants disclosed herein are conservative amino acid
changes, i.e., substitutions of similarly charged or uncharged amino
acids. A conservative amino acid change involves substitution of one of a
family of amino acids which are related in their side chains. Naturally
occurring amino acids are generally divided into four families: acidic
(aspartate, glutamate), basic (lysine, arginine, histidine), non-polar
(alanine, valine, leucine, isoleucine, proline, phenylalanine,
methionine, tryptophan), and uncharged polar (glycine, asparagine,
glutamine, cystine, serine, threonine, tyrosine) amino acids.
Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly
as aromatic amino acids.

[0076]In a peptide or protein, suitable conservative substitutions of
amino acids are known to those of skill in this art and generally can be
made without altering a biological activity of a resulting molecule.
Those of skill in this art recognize that, in general, single amino acid
substitutions in non-essential regions of a polypeptide do not
substantially alter biological activity (see, e.g., Watson et al.
Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin/Cummings
Pub. Co., p. 224).

Such substitutions may be made in accordance with those set forth in TABLE
2 as follows:

[0077]Other substitutions also are permissible and can be determined
empirically or in accord with other known conservative (or
non-conservative) substitutions.

[0078]Variants of the Herstatin and/or RBD Int8 polypeptide disclosed
herein include glycosylated forms, aggregative conjugates with other
molecules, and covalent conjugates with unrelated chemical moieties
(e.g., pegylated molecules). Covalent variants can be prepared by linking
functionalities to groups which are found in the amino acid chain or at
the N- or C-terminal residue, as is known in the art. Variants also
include allelic variants, species variants, and muteins. Truncations or
deletions of regions which do not affect functional activity of the
proteins are also variants.

[0079]A subset of mutants, called muteins, is a group of polypeptides in
which neutral amino acids, such as serines, are substituted for cysteine
residues which do not participate in disulfide bonds. These mutants may
be stable over a broader temperature range than native secreted proteins
(Mark et al., U.S. Pat. No. 4,959,314).

[0081]It is reasonable to expect that an isolated replacement of a leucine
with an isoleucine or valine, an aspartate with a glutamate, a threonine
with a serine, or a similar replacement of an amino acid with a
structurally related amino acid will not have a major effect on the
biological properties of the resulting secreted protein or polypeptide
variant. Properties and functions of Herstatin and/or RBD Int8
polypeptide protein or polypeptide variants are of the same type as a
protein comprising the amino acid sequence encoded by the nucleotide
sequences shown in SEQ ID NO:1 or 2, although the properties and
functions of variants can differ in degree.

[0082]Herstatin and/or RBD Int8 polypeptide variants include glycosylated
forms, aggregative conjugates with other molecules, and covalent
conjugates with unrelated chemical moieties (e.g., pegylated molecules).
Herstatin and/or RBD Int8 polypeptide variants also include allelic
variants (e.g., polymorphisms), species variants, and muteins.
Truncations or deletions of regions which do not preclude functional
activity of the proteins are also variants. Covalent variants can be
prepared by linking functionalities to groups which are found in the
amino acid chain or at the N- or C-terminal residue, as is known in the
art.

[0083]It will be recognized in the art that some amino acid sequence of
the Herstatin and/or RBD Int8 polypeptides of the invention can be varied
without significant effect on the structure or function of the protein.
If such differences in sequence are contemplated, it should be remembered
that there are critical areas on the protein which determine activity. In
general, it is possible to replace residues that form the tertiary
structure, provided that residues performing a similar function are used.
In other instances, the type of residue may be completely unimportant if
the alteration occurs at a non-critical region of the protein. The
replacement of amino acids can also change the selectivity of binding to
cell surface receptors (Ostade et al., Nature 361:266-268, 1993). Thus,
the Herstatin and/or RBD Int8 polypeptides of the present invention may
include one or more amino acid substitutions, deletions or additions,
either from natural mutations or human manipulation.

[0084]Of particular interest are substitutions of charged amino acids with
another charged amino acid and with neutral or negatively charged amino
acids. The latter results in proteins with reduced positive charge to
improve the characteristics of the disclosed protein. The prevention of
aggregation is highly desirable. Aggregation of proteins not only results
in a loss of activity but can also be problematic when preparing
pharmaceutical formulations, because they can be immunogenic (Pinckard et
al., Clin. Exp. Immunol. 2:331-340, 1967; Robbins et al., Diabetes
36:838-845, 1987; Cleland et al., Crit. Rev. Therapeutic Drug Carrier
Systems 10:307-377, 1993).

[0085]Amino acids in the Herstatin and/or RBD Int8 polypeptides of the
present invention that are essential for function can be identified by
methods known in the art, such as site-directed mutagenesis or
alanine-scanning mutagenesis (Cunningham and Wells, Science
244:1081-1085, 1989). The latter procedure introduces single alanine
mutations at every residue in the molecule. The resulting mutant
molecules are then tested for biological activity such as binding to a
natural or synthetic binding partner. Sites that are critical for
ligand-receptor binding can also be determined by structural analysis
such as crystallization, nuclear magnetic resonance or photoaffinity
labeling (Smith et al., J. Mol. Biol. 224:899-904, 1992 and de Vos et al.
Science 255:306-312,1992).

[0086]As indicated, changes are preferably of a minor nature, such as
conservative amino acid substitutions that do not significantly affect
the folding or activity of the protein. Of course, the number of amino
acid substitutions a skilled artisan would make depends on many factors,
including those described above. Generally speaking, the number of
substitutions for any given Herstatin and/or RBD Int8 polypeptide will
not be more than 50, 40, 30, 25, 20, 15, 10, 5 or 3.

[0087]In addition, pegylation of Herstatin and/or RBD Int8 polypeptides
and/or muteins is expected to provide such improved properties as
increased half-life, solubility, and protease resistance. Pegylation is
well known in the art.

Fusion Proteins

[0088]Fusion proteins comprising proteins or polypeptide fragments of
Herstatin and/or RBD Int8 polypeptide can also be constructed. Fusion
proteins are useful for generating antibodies against amino acid
sequences and for use in various targeting and assay systems. For
example, fusion proteins can be used to identify proteins which interact
with a Herstatin and/or RBD Int8 polypeptide of the invention or which
interfere with its biological function. Physical methods, such as protein
affinity chromatography, or library-based assays for protein-protein
interactions, such as the yeast two-hybrid or phage display systems, can
also be used for this purpose. Such methods are well known in the art and
can also be used as drug screens. Fusion proteins comprising a signal
sequence can be used.

[0089]A fusion protein comprises two protein segments fused together by
means of a peptide bond. Amino acid sequences for use in fusion proteins
of the invention can be utilize the amino acid sequence shown in SEQ ID
NOS:1 or 2 or can be prepared from biologically active variants of SEQ ID
NOS:1 or 2, such as those described above. The first protein segment can
include of a full-length Herstatin and/or RBD Int8 polypeptide.

[0090]Other first protein segments can consist of about 50 to about 79
contiguous amino acids from SEQ ID NO:1, or, with respect to SEQ ID NO:2,
from about 80 to 419 contiguous residues in length, wherein the
C-terminal 79 contiguous amino acids of SEQ ID NO:2 are present, or from
about 350 to 419 contiguous residues in length wherein the C-terminal 79
contiguous amino acids of SEQ ID NO:2 are present.

[0092]These fusions can be made, for example, by covalently linking two
protein segments or by standard procedures in the art of molecular
biology. Recombinant DNA methods can be used to prepare fusion proteins,
for example, by making a DNA construct which comprises a coding region
for the protein sequence of SEQ ID NOS:1 or 2 in proper reading frame
with a nucleotide encoding the second protein segment and expressing the
DNA construct in a host cell, as is known in the art. Many kits for
constructing fusion proteins are available from companies that supply
research labs with tools for experiments, including, for example, Promega
Corporation (Madison, Wis.), Stratagene (La Jolla, Calif.), Clontech
(Mountain View, Calif.), Santa Cruz Biotechnology (Santa Cruz, Calif.),
MBL International Corporation (MIC; Watertown, Mass.), and Quantum
Biotechnologies (Montreal, Canada; 1-888-DNA-KITS).

Cell Targeting

[0093]According to additional preferred aspects of the present invention,
cell surface receptor isoforms such as Herstatin- and/or RBD Int8
polypeptide-based agents can be used to target insulin receptor (IR) on
cells (e.g., insulin-resistant cells, IR-expressing cells involved with
some aspect of glucose regulation or metabolism, cancer cells, etc.).
Herstatin- and/or RBD Int8 polypeptide-based agents can be used to
deliver a locally acting biological agent that will affect the targeted
cell.

[0094]IR, in the context of the inventive targeting, is expressed on the
surface of cells and is accessible (specifically, or in addition to at
least one of the other known Herstatin targets: EGFR; HER-2; HER-3;
HER-4, ΔEGFR and IGF-IR) to exogenous molecules. For example, where
IR is present at higher levels on particular IR-bearing cells (e.g.,
adipocytes, hepatocytes, skeletal muscle cells, pancreatic beta cells,
brain/nerve cells, etc) as compared to other cells, they can be utilized
as preferential targets for systemic Herstatin- and/or RBD Int8
polypeptide-based agents and therapies. The differential expression of
the target receptor (e.g., IR) enables the specificity of Herstatin-
and/or RBD Int8 polypeptide-based agents-based therapy. Herstatin- and/or
RBD Int8 polypeptide-based agents (e.g., drugs, cytoxic agents, labeling
agents, etc.) directed against the target receptor preferentially affect
the targeted cell over normal tissue. For example, a Herstatin- or RBD
Int8 polypeptide-drug conjugate that binds a IR present predominantly on
particular cells (e.g., adipocytes, hepatocytes, skeletal muscle cells,
pancreatic beta cells, brain/nerve cells, etc) would be expected to
selectively affect those cells within a treated individual. Preferably,
the target receptor is accessible to the Herstatin- and/or RBD Int8
polypeptide-based agent, and is found in substantially greater
concentrations on the targeted cells (e.g., adipocytes, hepatocytes,
skeletal muscle cells, pancreatic beta cells, brain/nerve cells, etc)
relative to other cells that don't express IR or that express IR at
relatively low levels.

[0102]Toxins can also be targeted to specific cells by incorporation of
the toxin into Herstatin- and/or RBD Int8 polypeptide-coated liposomes.
The Herstatin- and/or RBD Int8 polypeptide-based agent directs the
liposome to the target cell where the bioactive compound is released. For
example, cytotoxins in Herstatin- and/or RBD Int8 polypeptide-coated
liposomes are used to treat cancer. In alternate embodiments, these
targeted liposomes are loaded with DNA encoding bioactive polypeptides
(e.g., inducible nitric oxide synthase; Khare et al. 2001).

[0103]Prodrugs or enzymes can also be delivered to targeted cells by
specific Herstatin- and/or RBD Int8 polypeptide-agents. In this case the
Herstatin conjugate consists of a Herstatin- and/or RBD Int8
polypeptide-based agent coupled to a drug that can be activated once the
polypeptide agent binds the target cell. Examples of this strategy using
antibodies have been reviewed (Denny 2001; Xu and McLeod 2001).

[0105]The specificity and high affinity of the Herstatin- and/or RBD Int8
polypeptide-based agents makes them ideal candidates for delivery of
toxic agents to a specific subset of cellular targets. Preferably, one or
more target receptors (e.g., IR, EGFR (HER-1, erbB-1); HER-2 (erbB-2);
HER-3 (erbB-3); HER-4 (erbB-4), ΔEGFR or IGF-IR) are present at
higher levels on the target cells (e.g., cancer, tumor cells) than on
non-cancer cells.

[0106]As used herein, a composition refers to any mixture. It can be a
solution, a suspension, liquid, powder, a paste, aqueous, non-aqueous or
any combination thereof.

[0107]As used herein, a combination refers to any association between or
among two or more items. The combination can be two or more separate
items, such as two compositions or two collections, can be a mixture
thereof, such as a single mixture of the two or more items, or any
variation thereof.

[0108]As used herein, a pharmaceutical effect refers to an effect observed
upon administration of an agent intended for treatment of a disease or
disorder or for amelioration of the symptoms thereof.

[0109]As used herein, treatment means any manner in which the symptoms of
a condition, disorder or disease or other indication, are ameliorated or
otherwise beneficially altered.

[0110]As used herein therapeutic effect means an effect resulting from
treatment of a subject that alters, typically improves or ameliorates the
symptoms of a disease or condition or that cures a disease or condition.
A therapeutically effective amount refers to the amount of a composition,
molecule or compound which results in a therapeutic effect following
administration to a subject.

[0111]In particular aspects, a therapeutic effect may also encompass
prophylaxis of symptoms of a condition.

[0112]As used herein, the term "subject" refers to animals, including
mammals, such as human beings. As used herein, a patient refers to a
human subject.

[0113]As used herein, the phrase "associated with" or "characterized by"
refers to certain biological aspects such as expression of a receptor or
signaling by a receptor that occurs in the context of a disease or
condition. Such biological aspects may or may not be causative or
integral to the disease or condition but merely an aspect of the disease
or condition.

[0114]As used herein, a biological activity refers to a function of a
polypeptide including but not limited to complexation, dimerization,
multimerization, receptor-associated kinase activity, receptor-associated
protease activity, phosphorylation, dephosphorylation,
autophosphorylation, ability to form complexes with other molecules,
ligand binding, catalytic or enzymatic activity, activation including
auto-activation and activation of other polypeptides, inhibition or
modulation of another molecule's function, stimulation or inhibition of
signal transduction and/or cellular responses such as cell proliferation,
migration, differentiation, and growth, degradation, membrane
localization, membrane binding, and oncogenesis. A biological activity
can be assessed by assays described herein and by any suitable assays
known to those of skill in the art, including, but not limited to in
vitro assays, including cell-based assays, in vivo assays, including
assays in animal models for particular diseases.

Pharmaceutical Compositions and Therapeutic Uses

[0115]Pharmaceutical compositions of the invention comprise a cell surface
receptor isoform such as Herstatin and/or RBD Int8 polypeptides, or
Herstatin- and/or RBD Int8 polypeptide-based agents of the claimed
invention in a therapeutically effective amount. The term
"therapeutically effective amount" as used herein refers to an amount of
a therapeutic agent to treat, ameliorate, or prevent a desired disease or
condition, or to exhibit a detectable therapeutic or preventative effect.
The effect can be detected by, for example, chemical markers or antigen
levels. Therapeutic effects also include reduction in physical symptoms.
The precise effective amount for a subject will depend upon the subject's
size and health, the nature and extent of the condition, and the
therapeutics or combination of therapeutics selected for administration.
Thus, it is not useful to specify an exact effective amount in advance.
However, the effective amount for a given situation is determined by
routine experimentation and is within the judgment of the clinician. For
purposes of the present invention, an effective dose will generally be
from about 0.01 mg/kg to 50 mg/kg or 0.05 mg/kg to about 10 mg/kg of the
Herstatin and/or RBD Int8 polypeptide constructs in the individual to
which it is administered. A non-limiting example of a pharmaceutical
composition is a composition that either enhances or diminishes signaling
mediated by the inventive target receptors (e.g., IR, EGFR, HER-2, HER-3,
ΔEGFR, HER-4 and IGF-IR). Where such signaling modulates a
disease-related process, modulation of the signaling would be the goal of
the therapy.

[0116]A pharmaceutical composition can also contain a pharmaceutically
acceptable carrier. The term "pharmaceutically acceptable carrier" refers
to a carrier for administration of a therapeutic agent, such as
antibodies or a polypeptide, genes, and other therapeutic agents. The
term refers to any pharmaceutical carrier that does not itself induce the
production of antibodies harmful to the individual receiving the
composition, and which can be administered without undue toxicity.
Suitable carriers can be large, slowly metabolized macromolecules such as
proteins, polysaccharides, polylactic acids, polyglycolic acids,
polymeric amino acids, amino acid copolymers, and inactive virus
particles. Such carriers are well known to those of ordinary skill in the
art. Pharmaceutically acceptable carriers in therapeutic compositions can
include liquids such as water, saline, glycerol and ethanol. Auxiliary
substances, such as wetting or emulsifying agents, pH buffering
substances, and the like, can also be present in such vehicles.
Typically, the therapeutic compositions are prepared as injectables,
either as liquid solutions or suspensions; solid forms suitable for
solution in, or suspension in, liquid vehicles prior to injection can
also be prepared. Liposomes are included within the definition of a
pharmaceutically acceptable carrier. Pharmaceutically acceptable salts
can also be present in the pharmaceutical composition, e.g., mineral acid
salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and
the like; and the salts of organic acids such as acetates, propionates,
malonates, benzoates, and the like. A thorough discussion of
pharmaceutically acceptable excipients is available in Remington's
Pharmaceutical Sciences (Mack Pub. Co., New Jersey, 1991).

Delivery Methods

[0117]Once formulated, the compositions of the invention can be
administered (as proteins/polypeptides, or in the context of expression
vectors for gene therapy) directly to the subject or delivered ex vivo,
to cells derived from the subject (e.g., as in ex vivo gene therapy).
Direct delivery of the compositions will generally be accomplished by
parenteral injection, e.g., subcutaneously, intraperitoneally,
intravenously or intramuscularly, myocardial, intratumoral, peritumoral,
or to the interstitial space of a tissue. Other modes of administration
include oral and pulmonary administration, suppositories, and transdermal
applications, needles, and gene guns or hyposprays. Dosage treatment can
be a single dose schedule or a multiple dose schedule.

[0118]Methods for the ex vivo delivery and reimplantation of transformed
cells into a subject are known in the art and described in, for example,
International Publication No. WO 93/14778. Examples of cells useful in ex
vivo applications include, for example, stem cells, particularly
hematopoetic, lymph cells, macrophages, dendritic cells, or tumor cells.
Generally, delivery of nucleic acids for both ex vivo and in vitro
applications can be accomplished by, for example, dextran-mediated
transfection, calcium phosphate precipitation, polybrene mediated
transfection, protoplast fusion, electroporation, encapsulation of the
polynucleotide(s) in liposomes, direct microinjection of the DNA into
nuclei, and viral-mediated, such as adenovirus (and adeno-associated
virus) or alphavirus, all well known in the art.

[0119]In a preferred embodiment, certain disorders (e.g., of
proliferation, such as cancer, etc), can be amenable to treatment by
administration of a therapeutic agent based on the provided
polynucleotide or corresponding polypeptide. The therapeutic agent can be
administered in conjunction with one or more other agents including, but
not limited to, receptor-specific antibodies and/or other agents (e.g.,
insulin-sensitizing agents, chemotherapeutic agents, etc). Administered
"in conjunction" includes administration at the same time, or within 1
day, 12 hours, 6 hours, one hour, or less than one hour, as the other
therapeutic agent(s). The compositions may be mixed for
co-administration, or may be administered separately by the same or
different routes.

[0120]The dose and the means of administration of the inventive
pharmaceutical compositions are determined based on the specific
qualities of the therapeutic composition, the condition, age, and weight
of the patient, the progression of the disease, and other relevant
factors. For example, administration of polynucleotide therapeutic
compositions agents of the invention includes local or systemic
administration, including injection, oral administration, particle gun or
catheterized administration, and topical administration. The therapeutic
polynucleotide composition can contain an expression construct comprising
a promoter operably linked to a polynucleotide encoding, for example,
about 80 to 419 (or about 350 to 419) contiguous amino acids of SEQ ID
NO:2. Various methods can be used to administer the therapeutic
composition directly to a specific site in the body. For example, an
abnormal tissue, or small metastatic lesion is located and the
therapeutic composition injected several times in several different
locations within the body of the tissue, or tumor. Alternatively,
arteries which serve a tissue or tumor are identified, and the
therapeutic composition injected into such an artery, in order to deliver
the composition directly into the tumor. A tissue or tumor that has a
necrotic center is aspirated and the composition injected directly into
the now empty center of the tissue or tumor. X-ray imaging is used to
assist in certain of the above delivery methods.

[0122]For gene therapy, therapeutic compositions containing a
polynucleotide are administered in a range of about 100 ng to about 200
mg of DNA for local administration in a gene therapy protocol.
Concentration ranges of about 500 ng to about 50 mg, about 1 mg to about
2 mg, about 5 mg to about 500 mg, and about 20 mg to about 100 mg of DNA
can also be used during a gene therapy protocol. Factors such as method
of action (e.g., for enhancing or inhibiting levels of the encoded gene
product) and efficacy of transformation and expression are considerations
which will affect the dosage required for ultimate efficacy of the
subgenomic polynucleotides. Where greater expression is desired over a
larger area of tissue, larger amounts of subgenomic polynucleotides or
the same amounts re-administered in a successive protocol of
administrations, or several administrations to different adjacent or
close tissue portions of, for example, a tumor site, may be required to
affect a positive therapeutic outcome. In all cases, routine
experimentation in clinical trials will determine specific ranges for
optimal therapeutic effect.

[0123]The therapeutic polynucleotides and polypeptides of the present
invention can be delivered using gene delivery vehicles. The gene
delivery vehicle can be of viral or non-viral origin (see generally,
Jolly, Cancer Gene Therapy (1994) 1:51; Kimura, Human Gene Therapy (1994)
5:845; Connelly, Human Gene Therapy (1995) 1:185; and Kaplitt, Nature
Genetics (1994) 6:148). Expression of such coding sequences can be
induced using endogenous mammalian or heterologous promoters. Expression
of the coding sequence can be either constitutive or regulated.

[0126]Further non-viral delivery suitable for use includes mechanical
delivery systems such as the approach described in Woffendin et al.,
Proc. Natl. Acad. Sci. USA 91(24):11581 (1994). Moreover, the coding
sequence and the product of expression of such can be delivered through
deposition of photopolymerized hydrogel materials or use of ionizing
radiation (see, e.g., U.S. Pat. No. 5,206,152 and WO 92/11033). Other
conventional methods for gene delivery that can be used for delivery of
the coding sequence include, for example, use of hand-held gene transfer
particle gun (see, e.g., U.S. Pat. No. 5,149,655); use of ionizing
radiation for activating transferred gene (see, e.g., U.S. Pat. No.
5,206,152 and WO 92/11033).

Conditions Treatable

[0127]Particular aspects of the present invention, for the first time,
disclose that Herstatin or Int8 RBD polypeptides, and variants thereof,
can not only modulate the expression/level of cellular insulin receptors
(IR) (both pro-IR and IR), but also modulate IR-mediated signal
transduction (e.g., ERK pathway). According to particular aspects,
Herstatin or Int8 RBD polypeptides, and variants thereof can be used in
therapeutic methods and pharmaceutical compositions to treat a variety of
conditions having an aspect related to, or associated with altered IR
expression or altered IR-mediated signaling at a cellular level. Such
methods comprising administering to a subject having such a condition, a
therapeutically effective amount of a Herstatin or Int8 RBD polypeptide,
or a variant thereof, that binds to the extracellular domain of cellular
target insulin receptor. Such methods also encompass gene
delivery-related methods.

[0128]IR is well known in the art to be involved with, inter alia,
glycemic control (e.g., hyper- and hypo-glycemia) and glucose metabolism.
Accordingly, conditions having an aspect related to, or associated with
altered glycemic control and/or glucose metabolism are within the scope
of treatable conditions according to the present invention. Such
conditions include, but are not limited to insulin resistance syndrome,
pre-diabetic conditions, metabolic syndrome, type 1 and type 2 diabetes,
cardiac disease, diabetes-associated vascular disease, atherosclerosis,
hypertension, diabetes-associated lipid metabolism disorders
(dyslipidemia), obesity, critical illness, neurodegenerative disorders,
and combinations thereof.

[0129]Insulin resistance syndrome has become the major health problem of
our times, and is associated with obesity, dyslipidemia, atherosclerosis,
hypertension, and type-2 diabetes shorten life spans, and
hyperandrogenism with polycystic ovarian syndrome affect quality of life
and fertility in increasing numbers of women (see, e.g., Ten & Maclaren,
J. Clin Endocrinol Metab., 89:2526-2539, 2004; and see Le Roith 7 Zick,
Diabetes Care 24:588-597, 2001; both incorporated herein by reference).
In particular preferred aspects, Herstatin or Int8 RBD polypeptide, or
variants thereof can be used to treat insulin resistance syndrome.

[0130]Insulin resistance and associated abnormalities are believed to have
a role in pregnancy induced hypertension (new-onset hypertension), and
many features of the insulin resistance syndrome are associated with this
condition (see, e.g., Seely & Solomon, J. Clin. Endocrinol. Metab.,
88:2393-2398, 2003; incorporated herein by reference). According to the
present invention, Herstatin or Int8 RBD polypeptide, or variants thereof
can be used to treat hypertension and new-onset hypertension.

[0131]In prolonged critical illness neuroendocrine changes lead to more
extensive metabolic changes. For example, insulin resistance and
hyperglycemia are associated with critical illness (e.g., in surgically
critically ill populations with or without diabetes, post-myocardial
infarction in patients with diabetes, etc.) (see, e.g., Ronbinson & H.
van Soeren, AACN Clinical Issues, 15:45-62, 2004; incorporated herein by
reference). According to the present invention, Herstatin or Int8 RBD
polypeptide, or variants thereof can be used to treat critical illness.

[0132]Significantly, impairment of insulin signaling in the brain has been
linked, on the basis of studies using IR-knockout (NIRKO) mice, to
neurodegenerative diseases. NIRKO mice exhibit a complete loss of
insulin-mediated activation of phosphatidylinositol 3-kinase and
insulin-mediated inhibition of neuronal apoptosis, resulting in markedly
reduced phosphorylation of Akt and GSK3 β and leading to a
substantially increased phosphorylation of the microtubule-associated
protein Tau, a hallmark of neurodegenerative diseases (e.g., Alzheimer's
disease) (see, e.g., Schubert et al., PNAS 101:3100-3105, 2004,
incorporated herein by reference). According to the present invention,
Herstatin or Int8 RBD polypeptide, or variants thereof can be used to
treat to neurodegenerative diseases (e.g., Alzheimer's disease).

[0134]For example, the inventive treatment methods may further comprise
administering a therapeutically effective amount of a receptor-specific
antibody that binds to the extracellular domain of a target receptor
selected from the group consisting of: IR, EGFR (HER-1, erbB-1);
εEGFR; HER-2 (erbB-2); HER-3 (erbB-3); HER-4 (erbB-4), and
IGF-IR.

[0135]Alternatively, the inventive treatment methods may further comprise
administering a therapeutically effective amount of an agent selected
from the group consisting of: insulin, insulin-sensitizing agents,
insulin secretogogues, and combinations thereof. Preferably, the
insulin-sensitizing agent is selected from the group consisting of
biguanides, metformin, thiazolidinediones (glitazones), and combinations
thereof. Preferably, the insulin secretogogue is selected from the group
consisting of sulfonylureas, meglitinides, and combinations thereof (see,
e.g., Zangeneh et al., Mayo Clin Proc., 78:471-479, 2003, incorporated by
reference herein).

[0136]The present invention will now be illustrated by reference to the
following examples which set forth particularly advantageous embodiments.
However, it should be noted that these embodiments are illustrative and
are not to be construed as restricting the claimed invention in any way.

[0143]The dissociation constant (KD) and maximal binding (Bmax)
of Herstatin were determined by nonlinear regression analysis of the plot
of pmol of bound versus nM of Herstatin added. Statistical comparisons
between different binding curves were performed by extra sums-of-squares
F-test nonlinear regression coefficients. All tests were performed
(α=0.05) using GraphPad® Prism 4® software (GraphPad®
Software, 1994-2003).

Pull-down Assays with Int8 Peptide Immobilized on Protein S Agarose:

[0144]About 100 μl of a 50% suspension of S-protein agarose (Novagen)
is incubated with or without 100 μg of int8 peptide with an S-protein
tag, at room temperature for 1 hr, and then washed twice with 500 μl
PBS. The agarose samples are then incubated at room temperature for 1 hr
with 200 μg of transfected cell extract, then washed twice with 500
μl of PBS with 1% NP40. The proteins associated with the resin are
eluted at 92° C. for 2 min in 40 μl of SDS-sample buffer, and
analyzed as a Western blot.

Growth Assays:

[0145]Cells (4×104) were plated in quadruplicate in 24-well
plates, incubated in serum-free DMEM for 24 hours, and treated with
either 10 nM insulin (Sigma) or an equivalent volume of vehicle (25 mM
HEPES). At the indicated time points, cell monolayers were washed with
PBS and incubated for 30 minutes at 37° C. with 30 μl of MTS
reagent [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sul-
fophenyl-2H-tetrazolium) inner salt Aqueous One Solution (Promega;
Madison, Wis.) dissolved in 270 ml PBS] per well. Absorbance at 490 nm
was determined a Bio-Tek plate reader.

EGFR Inhibitor Studies

[0146]Control MCF-7 cells were serum-starved overnight and treated with
the EGFR kinase inhibitor AG1478 (Sigma) or vehicle (DMSO) for 5 minutes
prior to the addition of 14 nM EGF or 10 nM insulin (Sigma). After growth
factor treatment, cell lysates were prepared and analyzed for ERK and
Akt/PKB activation as described above. The 24-hour treatment was done in
regular growth medium.

[0147]The interaction of Herstatin with IR in transfected 3T3 cells
(IRA-3T3) was investigated. Herstatin bound specifically to IR at nM
concentrations, and IR was thus shown herein to be a target of Herstatin.

[0153]Results. FIG. 2 shows that Herstatin expression not only
up-regulated IR expression (including pro-IR), but also up-regulated IR
activation (and thus signaling) in MCF-7 cells. Control and
Herstatin-expressing MCF-7 cells were grown in complete medium prior to
an overnight incubation in serum-free medium. Insulin was then added to
the control and Herstatin-expressing cells and whole-cell lysates were
prepared at the indicated times and processed directly for Western
immunoblots with anti-insulin receptor (IR), phospho-Akt, Akt,
phospho-ERK, and ERK antibodies, or first immunoprecipitated with anti-IR
antibody and immunoprecipitates (IP) then analyzed by Western
immunoblotting with anti-phosphotyrosine and anti-IR antibodies after
transfer to nitrocellulose membranes. Following incubation of blots with
primary antibodies, immunoreactive proteins were detected by enhanced
chemiluminescence after a secondary incubation with HRP-conjugated
secondary antisera. Similar results were obtained with a second
Herstatin-expressing MCF-7 clone.

[0156]The effect of Herstatin expression on insulin-stimulated ERK
activation/signaling was further investigated.

[0157]Methods. Methods were as described above under EXAMPLE III herein
above.

[0158]Results. FIG. 3 shows, in MCF-7 cells, that Herstatin expression
amplified insulin-stimulated ERK activation. Control and
Herstatin-expressing MCF-7 cells were treated and analyzed as those of
FIG. 2. Film exposures of enhanced chemiluminescence signals were
quantified by scanning densitometry, and the values for the phospho-ERK
signals were normalized to the ERK signals to determine the relative
level of ERK phosphorylation as a measure of activation.

[0161]This is because the MEK (MAPK kinase)-ERK pathway has been shown to
be significantly involved in glucose transport (e.g., Harmon et al., Am.
J. Physiol. Endocrinol. Metab., 287:E758-E766, 2004). Specifically,
Harmon et al show specific inhibition of MAPK kinase (MEK) by the
inhibitors PD-98059 and U-0216, resulting in significant inhibition of
insulin-stimulated glucose uptake. The data support the importance of MEK
for activation of GLUT4, and further, since the only target of MEK is
ERK, the importance of the MEK (MAPK kinase)-ERK pathway for glucose
transport.

EXAMPLE V

Herstatin Altered the Expression of an Array of Proteins that are Directly
Involved in Insulin Action

[0162]In addition to the regulation of insulin receptor protein, the
regulation of the IRS-1 and IRS-2 proteins and Shc (that function as
adapter proteins linking the activated insulin receptor to some of its
downstream pathways), the expression of ERK and Akt/PKB, and the
regulation of the IGF-IR (which may contribute to enhanced insulin
receptor activation by decreasing the proportion of insulin
receptor/IGF-I receptor hybrids, which do not respond to insulin) was
investigated.

[0164]Results. FIG. 4 shows that Herstatin altered the expression of an
array of proteins that are directly involved in insulin action. Lysates
from control and Herstatin-expressing MCF-7 cells were prepared from
respective untreated (no insulin) cells following overnight incubation in
serum-free media, and processed directly or (in the case of the IR) also
immunoprecipitated prior to Western immunoblot analysis as described in
relation to FIG. 2.

[0165]These data illustrate that Herstatin: up-regulates insulin receptor
protein as assessed by direct Western immunoblot and following
immunoprecipitation; mediates the apparent phosphorylation state of the
IRS-1 and IRS-2 (differentially down-regulated compared with IRS-1)
proteins that function as adapter proteins linking the activated insulin
receptor to some of its downstream pathways (see, e.g., Le Roith 7 Zick,
Diabetes Care 24:588-597, 2001, discussing role of IRS (IR substrate)
proteins in IR-mediated signal transduction); elicits a slight decrease
in IRS-2 expression; alters the relative expression of Shc isoforms
expressed; increases the relative expression ratio of ERK1 and ERK2; and
down-regulates the IGF-IR, which may contribute to enhanced insulin
receptor activation by decreasing the proportion of IR/IGF-IR hybrids,
which do not respond to insulin.

EXAMPLE VI

The EGFR inhibitor AS1478 does not Affect Insulin Signaling or Lead to an
Increase in IR

[0166]FIG. 5 shows, according to particular aspects, that the EGFR
inhibitor AS1478 did not affect insulin signaling.

[0167]FIG. 6 shows, according to particular aspects, that inhibition of
the EGF receptor with an EGF receptor-specific inhibitor did not lead to
an increase in insulin receptor.